Procedure

Make a plasmid map of your design

This is key. You will want it for primer design, checking your primers, assessing sequencing reactions, etc. I use APE, open-source software. See my APE use page

Design primers

The primers should confer 20-100 bp of homology between to adjacent overlapping segments. 40 - 100 bp is ideal; substantially shorter or longer will give you lower yields.

The annealing portion of the primer should have Tm between 62oC and 65oC as calculated by this Finnzymes website

This formula is applicable to Phusion DNApolymerase, the DNA polymerase used to form the DNA you will assemble.

Use cheap primers

If ordering with IDT, primers should be 60 bp if you are encoding homology. The price per base pair jumps when you add the 61st base pair: we pay ~$9 for a 60 bp primer but ~ $34 for a 61 bp primer. Using less than 60 bp reduces the length of the homolgy between adjacent DNA pieces in the assembly. Note: there are cases when you use standard size (18-22 bp) primers as is discussed in this page. *** DISCUSS ***

Generate PCR fragments

Purify PCR fragments

Gibson assembly reaction

Transformation

Sequencing

Examples

you can chose where the seam is if you use longer oligos

Dpn1

RFP for backbone: don't screen red colonies!

Making your own Gibson mix

Recipe

Tips:

Balancing the ratio of T5 & Phusion is mportant given the mechanism. The exonuclease is so concentrated relative to the desired concentration in the mix that it should be diluted 10X before use.